Neural Regen Res. 2016 Jan; 11(1): 49–52.
doi: 10.4103/1673-5374.169628
PMCID: PMC4774222
Tracking of iron-labeled human neural stem cells
by magnetic resonance imaging in cell replacement
therapy for Parkinson's disease
Milagros Ramos-Gómez1,2 and Alberto Martínez-Serrano, Ph.D.3,*
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Abstract
Human neural stem cells (hNSCs) derived from the ventral mesencephalon are powerful research tools and
candidates for cell therapies in Parkinson's disease. However, their clinical translation has not been fully
realized due, in part, to the limited ability to track stem cell regional localization and survival over long
periods of time after in vivo transplantation. Magnetic resonance imaging provides an excellent non-invasive
method to study the fate of transplanted cells in vivo. For magnetic resonance imaging cell tracking, cells
need to be labeled with a contrast agent, such as magnetic nanoparticles, at a concentration high enough to
be easily detected by magnetic resonance imaging. Grafting of human neural stem cells labeled with
magnetic nanoparticles allows cell tracking by magnetic resonance imaging without impairment of cell
survival, proliferation, self-renewal, and multipotency. However, the results reviewed here suggest that in
long term grafting, activated microglia and macrophages could contribute to magnetic resonance imaging
signal by engulfing dead labeled cells or iron nanoparticles dispersed freely in the brain parenchyma over
time.
Keywords: human neural stem cells, Parkinson's disease, magnetic resonance imaging, magnetic
nanoparticles, stem cell transplantation
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In Vivo Tracking of Human Neural Stem Cells
One of the goals of stem cell research is the in vitro and in vivo generation of neurons which could replace
the neurons lost in certain acute conditions and chronic diseases of the nervous system. Stem cell therapies
are promising strategies for the treatment of several neurodegenerative diseases, such as Parkinson's disease
(PD) characterized by a progressive loss of integrity, function and number of dopaminergic neurons (DAn)
present in the substantia nigra pars compacta (SNpc). Stem cells, and more specifically neural stem cells
(NSCs), constitute a viable option for regenerative medicine in PD, in particular for cell replacement
therapies (CRT) both in the pre-clinical and clinical experimental settings (Lindvall et al., 2004).
Previous clinical studies of cell replacement in PD were based on the transplantation of fresh human fetal
ventral mesencephalic (VM) tissue into the caudate and putamen of PD patients. Even when these studies
provided clear-cut proof-of-concept type of evidence of CRT efficacy, fetal tissue transplants will not reach
the status of a routine clinical practice due to practical and ethical issues. Indeed, some of those clinical
experiments provided mixed outcomes, an aspect that still needs to be clarified. Among the practical issues,
CRT for PD using fresh fetal tissue requires from six to seven human fetuses to provide enough cells for
transplantation of a single patient, there is an elevated cell death rate of the transplanted cells, a lack of
reproducibility between centers and the appearance of serious adverse side effects (dyskinesias) in some
patients.
A tremendous progress has been achieved in recent years in our understanding of stem cell biology. Stem
cells, in particular human induced pluripotent stem cells (hi-PSCs) and human NSCs (hNSCs) are today
considered as of potential help to circumvent the mentioned problems, and to constitute the best alternative
source of DAn (Lindvall et al., 2004). Stem cell therapies could overcome many of the issues associated
with primary fetal tissue, thus helping to bring forward CRT to become a reliable and effective treatment for
neurodegenerative disorders (Politis and Lindvall, 2012).
Recent in vitro and in vivo work has thus aimed at obtaining suitable sources of human NSCs with the
capacity to differentiate into DAn, in turn endowed with the required genuine properties of SNpc neurons
lost in PD. Previous studies with rodent and human DAn precursors indicated poor growth potential and
unstable phenotypical properties. In this regard, we have established a new immortalized cell line obtained
from human fetal ventral midbrain (VM) tissue and modified to express Bcl-XL, (hVM1-Bcl-XL or hVM, in
short) which shows a great potential for the generation of SNpc DAn in vitro (Courtois et al., 2010). Bcl-XL
allows for human fetal VM stem cells to stably generate mature SNpc DAn both in vitro and in vivo.
Mechanistically, Bcl-XL not only exerts the expected anti-apoptotic effect but also induces pro-neural and
dopamine-related transcription factors, resulting in a high yield of DAn with the correct phenotype for the
development of cell therapies for PD (Courtois et al., 2010).
Since functional maturation of human DAn in vivo requires long survival times, the behavioral amelioration
induced by the transplantation of hVM cells in the striatum of parkinsonian rats has been analyzed for up to
5 months, demonstrating an amelioration of spontaneous motor behavior and cognitive performance in
parkinsonian animals after receiving human VM neural stem cell grafts (Ramos-Moreno et al., 2012). VMderived cell lines did survive and integrated well from the second month after transplantation and mature
neurons were generated, but the morphology of many DAn in hVM cell transplants was still immature.
Electrophysiological maturation and integration has also been demonstrated in more recent short- to midterm transplantation experiments (Ramos-Moreno T. and Martinez-Serrano A., unpublished results). Thus,
future studies will be focused on determining the safety and therapeutic effect on biochemical,
electrophysiological and behavioral grounds of hVM cells at longer time points (up to 12 months).
Human NSCs derived from the VM region are thus powerful research tools and candidates for cell therapies
in PD. However, their clinical translation has not been fully realized due, in part, to the limited ability to
track stem cell regional localization and survival over long periods of time after in vivo transplantation. Until
now, behavioural tests and the subsequent histological analysis have been the preferred methods used to
evaluate graft viability, differentiation and effects of transplanted cells. Research in CRT requires noninvasive and sensitive imaging techniques to unequivocally track the localization of transplanted cells.
Tracking noninvasively the long-term spatial destination and final residence of transplanted cells in vivo, to
monitor their survival, migration, differentiation and regenerative impact, has become a critical
methodology in evaluating the efficacy of stem cell therapy procedures.
Magnetic resonance imaging (MRI), due to its inherent high spatial resolution, provides an excellent noninvasive method to study the fate of transplanted cells in vivo. To detect the transplanted cells by MRI with
high sensitivity, it is necessary to label them with magnetic nanoparticles (MNPs) prior to their implantation.
Labeling cells with MNPs has been shown to induce sufficient contrast for MRI visualization without
impairment of their functional properties, allowing the detection of even ~500 labeled cells (Kallur et al.,
2011). Therefore, MRI, in combination with other in vivo molecular imaging techniques, like positron
emission tomography (PET) or bioluminescence live imaging (BLI) can provide insight into important
grafting parameters such as optimal cell “dose”, localization and migration of the cells, cell survival and
proliferation kinetics, graft volume, and cell differentiation or de-differentiation patterns, which can aid
clinical implementation of cell therapy (Swijnenburg et al., 2007). To determine some of these parameters in
a reliable manner, for instance graft volume or cell proliferation, very precise titration controls are needed to
determine the detection limit of the MRI equipment.
For MRI cell tracking, cells need to be labeled with the contrast agent, such as MNPs, at a concentration
high enough to be easily detected by MRI. Most labeling techniques currently use one of the following
approaches: attaching magnetic nanoparticles to the stem cell surface or internalizing biocompatible
magnetic nanoparticles by endocytosis. Surface labeling tends to result in lower iron content per cell and
promotes a rapid reticuloendothelial recognition and clearance of labeled cells (Syková and Jendelová,
2005). Therefore, endocytosis of MNPs during in vitro cell cultivation is the preferred method to label cells
for in vivo tracking by MRI.
Since MNPs do not efficiently label either non-phagocytic or non-rapidly dividing mammalian cells in vitro,
in most studies, cells are co-incubated with MNPs in the presence of transfection agents (TAs), such us
poly-L-lysine or protamine sulfate, to improve their uptake by the cells. However, the use of TAs to label
cells might have harmful side effects. Since most TAs are toxic to cells producing a slight increase in
apoptosis shortly after labeling (Thu et al., 2009), they may reduce cell viability. This toxic effect is
probably due to the use of TAs and not to the MNPs themselves, since it disappeared at later stages of NSC
culture, while iron nanoparticles still remained inside the cells. Moreover, it has also been reported that
MNPs complexed with poly-L-lysine blocked the differentiation of human mesenchymal stem cells into
chondrocytes (Kostura et al., 2004). Therefore, the use of TAs remains problematic, although recent
evidence indicates that incubation of hNSCs with MNPs in the absence of TAs is not always ineffective,
rather the contrary (Ramos-Gómez et al., 2015). The labeling efficiency can be improved by prolonged
incubation times and elevated iron doses, resulting in a high number of internalized MNPs per cell (Figure
1). However, the use of relative high concentrations of MNPs to label hNSCs might be toxic or affect some
of their functional properties, causing alterations in their differentiation processes. Therefore, a careful
titration of MNP concentration is required to set optimal conditions resulting in efficient cell labeling
without impairment of cell survival, proliferation, self-renewal, and multipotency (Kallur et al., 2011;
Eamegdool et al., 2014; Ramos-Gómez et al., 2015). Thus, an extensive study of the properties of NSCs
labeled with MNPs must be carried out to identify the effects of MNPs on hNSCs biology. Upon loading,
cellular viability and differentiation, average distance of migration and changes in intracellular calcium ion
concentration have been analyzed in many studies to determine optimal loading conditions and the impact of
iron on cellular metabolism (Kallur et al., 2011; Eamegdool et al., 2014; Ramos-Gómez et al., 2015). These
studies have confirmed that MNPs-labeling of human NSCs showed no adverse effects on cell biology when
used at an optimized dosage. MNPs did not affect short-term survival, proliferation rates, multipotency or
differentiation capacity of NSCs. However, after long-term in vitro differentiation, some changes appeared
in both the proportion of neural phenotypes generated, as well as in the number of proliferating cells in some
of the labeled cultures when compared with non-labeled control cell cultures. MNP-labeled cell cultures
contained significantly less nestin- and GFAP-positive cells than the unlabeled controls. These effects have
been shown in hNSCs after 18 days in culture and could be caused by a delayed toxicity due to iron
overloading, which leads to cellular stress responses that may have effects on cell properties, manifested
only after longer experimental times (Kallur et al., 2011). Moreover, with a prolonged exposure, MNPs
translocated from the cytoplasm into the peri-nuclear region, and the migration capacity of hNSCs may be
affected (Eamegdool et al., 2014).
Figure 1
hVM-MNPs labeled cells.
In spite of the effects described when cells are cultured in the presence of MNPs for extended periods of
time, properly labeled hVM cells maintain their stemness and differentiation potential. Labeling hVM cells
with MNPs did not affect the percentage of tyroxyne hydroxilase positive cells, a typical dopaminergic
marker, obtained during the differentiation process. No significant changes in the percentage of nestin,
GFAP or β-III-tubulin hVM cells compared to MNPs-hVM cells were observed after 7 days of in vitro
differentiation (Ramos-Gómez et al., 2015). Similar findings were also obtained with the neuronal
transcription factor doublecortin, a marker of immature neurons. In addition, it was found that once MNPlabeled progenitor cells differentiated into astrocytes or neurons, they did not lose their MNP label (Shen et
al., 2013). Therefore, after optimization of the appropriate dose and incubation times, MNP-labeling of
human NSCs left the quantitative profile of the in vitro proliferation and differentiation capacity of the cells
unaltered. According to the literature, no differences in MNP-labeling have been described for different
sources of stem cells. Efficient labeling of hi-PSCs (Ruan et al., 2011) and embryonic stem cells (Parsa et
al., 2015) can be achieved with no negative effects on growth, morphology, viability, proliferation and
differentiation allowing noninvasive in vivo MRI tracking of the transplanted cells. In addition, in vivo MR
tracking of MNP-labeled bone marrow-derived NSCs after autologous transplantation can be accomplished
(Ke et al., 2009).
MRI can be used for tracking of MNP-labeled neural stem cells for studying the in vivo localization and
migration of grafted stem cells in longitudinal studies. It has been shown that labeling of hVM cells with
MNPs does not affect their overall biology and survival rate. No significant changes have been detected in
the phenotypes generated in vivo after transplantation in response to cell labeling (Eamegdool et al., 2014;
Ramos-Gómez et al., 2015). The MNPs-labeled hNSCs grafted into hemiparkinsonian rats can be
successfully visualized using MRI (Figure 2) at different time points, up to 5 months after transplantation,
supporting the use of commercial MNPs to track cells for short- and mid-term periods in vivo (RamosGómez et al., 2015). MNPs are detected by MRI as a hypointense signal which persisted for at least 5
months after transplantation. This hypointense signal, corresponding to the presence of MNPs, showed no
significant changes in both the location and volume over time (Kallur et al., 2011). However, of great
concern is the notion that activated microglia and macrophages will engulf dead labeled cells or iron
nanoparticles dispersed freely in the brain parenchyma, contributing thus to the MRI signal and probably
inducing a significant microglia proliferation (Cupaioli et al., 2014). Dead NSCs and the iron nanoparticles
expelled from them may be phagocytosed by activated microglia and macrophages that are present at the
NSC injection site over time (Ramos-Gómez et al., 2015). Thus, long-term MR images should be interpreted
with caution due to the possibility that some MNPs may be expelled from the transplanted cells and
internalized by host microglia cells. Among complementary aiding tools, the simultaneous use of BLI in the
same animals seems to be accurate enough (although resolution is lower) to confirm that the MRI signal is
coming from alive transplanted cells. Although several clinical trials using MNP-labeled cells have already
been performed, clinical application of MNP-cell tracking by MRI is still many years away (Bernsen et al.,
2015). However, much can already be learned from animal models which may help to elucidate the
mechanisms underlying the success of cell-based therapies.
Figure 2
MRI of hVM-MNP labeled cells transplanted into rat brain.
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Conclusions
To sum up, the ability to monitor the time course of survival, migration, and distribution of stem cells
following transplantation by MRI will provide critical information for optimizing future therapies based on
cell transplantation for several neurodegenerative diseases, such as Parkinson's disease. It has been
demonstrated that labeling hNSCs with MNPs, using optimal doses of iron, does not affect their overall
biology, survival rate and in vitro and in vivo differentiation patterns. Grafting of hNSC labeled with MNPs
allows cell tracking by MRI for long periods of time without showing significant neuronal or systemic
toxicities or behavioral changes. However, the results reviewed here suggest that in long term grafting,
activated microglia and macrophages could contribute to the MRI signal by engulfing dead labeled cells or
iron nanoparticles dispersed freely in the brain parenchyma over time. Thus, long-term MR images should
be interpreted with caution, and interpreted in combination with other information like that offered by in
vivo PET and BLI.
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Footnotes
Funding: Work at the author's laboratories was supported by: To AMS: Instituto de Salud Carlos-III
(RETICS TerCel RD12/0019/0013), Comunidad Autónoma de Madrid (S2010-BMD-2336), MINECO
(SAF2010-17167) and the institutional grant of the Fundación Ramón Areces to the CBMSO. To MRG:
Reina Sofia Foundation and Comunidad Autónoma Madrid (S2010-BMD-2460).
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Figure 1
hVM-MNPs labeled cells.
Fluorescence micrographs showing human ventral midbrain (hVM) cells labeled with magnetic
nanoparticles (MNPs) marked in green. Nuclei are shown in blue stained with Hoechst. Scale bar: 20 μm.
Figure 2
MRI of hVM-MNP labeled cells transplanted into rat brain.
Two consecutive in vivo magnetic resonance images of hVM cell suspensions (3 × 105 cells) transplanted
into the left (hVM cells, L) and right (hVM-MNP labeled cells, R) striata. MRI was performed eight weeks
after cell transplantation. hVM-MNP labeled cells can be easily detected in coronal T2*-weighted images as
dark hypointense signals in the area where the cells have been injected (arrows). No MRI signal was
detected when unlabeled hVM cells were transplanted (left striatum, L). Scale bar: 2 mm. hVM: Human
ventral midbrain; MNP: magnetic nanoparticle; MRI: magnetic resonance imaging; R: right stratum.